Immunoaffinity enrichment analysis of protein methylation

Protein methylation is a posttranslational modification that mostly occurs on arginine and lysine residues. When methyltransferases are knocked out in mice, it can result in embryonic lethality or early death, indicating the vital biological role this group of enzymes. The record of methylated peptide number identified in a single experiment is 249 arginine methylation sites.

Here, we have developed highly specific antibodies against arginine and lysine methylation. After immunoaffinity enrichment and mass spectrometry analysis, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and 160 lysine methylation sites in human cell line HCT116.

Firstly, we developed antibodies against methylated lysine, using a peptide library that had a single modified lysine in a peptide library containing amino acids at surrounding positions.

Next, we pull down the methylated peptide and analysis them with LC/MS/MS. And then we have classified the arginine methylation pattern form mouse brain and embryo.

At last, we have labeled the total protein with dimethylation in vitro. Use this artificial sample, we identified more than thousands of methylated peptide, however, the normal sample has fewer than 100 lysine methylation sites, suggesting that this modification is restricted to a small subset of proteins.

Methylation of arginine and lysine protein residues has been known of for many years, the challenge for the field is lacking of tools and methods for studying these modifications. This study provide a powerful tools to get insight into the field of protein methylation.

MC4R is a G-protein coupled receptor that binds?-melanocyte stimulating hormone (?-MSH). In murine models MC4 receptors have been found to be involved in feeding behavior, the regulation of metabolism, sexual behavior, and male erectile function. Both in rodents and humans, melanocortin-4 receptors (MC4Rs) suppress appetite and prevent obesity. Activation of melanocortin-4 receptors (MC4Rs) restrains feeding and prevents obesity.

We deleted and re-expressed MC4Rs from cre-expressing neurons and observed the phenotype. As the key MC4R-expressing neurons are glutamatergic, it is almost a certainty that they release glutamate and excite their downstream target neurons.

Next, we show that MC4Rs on glutamatergic neurons are necessary for preventing hyperphagia and obesity. Then, we have rebuild MC4Rs only in SIM1 neurons rescued?60% of the obesity and 100% of the hyperphagia of MC4R-null mice. This result indicates that MC4Rs on SIM1 neurons regulate feeding. Because the transcription factor SIM1 is required for PVH development.

To summary, we demonstrate that the MC4R-expressing neurons regulating feeding are SIM1+, located in the PVH, glutamatergic and not GABAergic, and do not express oxytocin, corticotrophin-releasing hormone and vasopressin. We identify downstream projections from these glutamatergic neurons to the lateral parabrachial nucleus, which could mediate the appetite suppressing effects.

Numerologist Warda is hooked on OG-L002 fishing, collecting. And lastly her encouragement comes from socializing along with her companions.